Combination of an agent that attenuates topoisomerase 1 activity and an agent that inhibits heat shock protein 90 for use in chemotherapy

ABSTRACT

The present invention relates to the use of a first agent that attenuates Topoisomerase I (Topo I) activity and a second agent that inhibits Heat Shock Protein 90 (HSP90) for use in chemotherapy. The agents are particularly useful in the treatment of cancer and destruction of micro-organisms. The invention also relates to screening methods, diagnostic methods and methods for evaluating or monitoring chemotherapy regimens.

The present invention relates to the treatment of medical conditionsusing a combination of chemotherapeutic agents.

In general, when chemotherapy is used for the treatment of human cancersand the like, a combination of agents is employed. In the past, thereasoning behind the choice of which particular combinations of agentsare used has been essentially a pragmatic decision, often based more ontolerances to toxicity rather than specific targets.

Recent studies of the process of carcinogenesis, have revealed that manyof the genetic lesions involved, cause errors in the cell division/deathpathways. The molecular changes that result from such lesions initiatethe cancer process. Due to this the molecules involved in such changesprovide potentially highly specific targets for chemotherapy. Using thetargets identified by this approach new therapeutic agents may beintroduced into the clinic. However, to achieve optimal clinical benefitfrom these agents, they may too need to be used in combination withother anticancer drugs. Again the choice of which particularcombinations of agents are used has been a decision based more ontolerances to toxicity rather than specific targets.

There is also a need to develop new and improved antimicrobial agents.Antibiotic resistance is a growing problem and there is an increasingneed to provide effective combination therapies.

According to a first aspect of the present invention, there is provideda use of a first agent that attenuates Topoisomerase I activity and asecond agent that inhibits Heat Shock Protein 90 activity for themanufacture of a medicament for contemporaneous or sequentialadministration in chemotherapy.

According to a second aspect of the present invention, there is provideda method for conducting chemotherapy comprising contemporaneously orsequentially administering to a person or animal in need of saidtreatment a therapeutically effective amount of a first agent thatattenuates Topoisomerase I activity and a second agent that inhibitsHeat Shock Protein 90 activity.

According to a third aspect of the present invention, there is provideda composition for use in chemotherapy comprising therapeuticallyeffective amounts of a first agent that attenuates Topoisomerase Iactivity and a second agent that inhibits Heat Shock Protein 90 activityand a pharmaceutically acceptable vehicle.

By “chemotherapy” we mean treatment of cells to cause a targeted celldeath. Chemotherapy is required in cancer treatment where it isdesirable to target transformed cells. Chemotherapy is also employed totreat infections caused by pathogens (e.g. bacterial, fungal or viralinfections).

Topoisomerase I (Topo I) is an enzyme that catalyzes the transport of asingle strand of DNA through another single strand of DNA. This works toalleviate the topological problems encountered by intracellular DNA.Topoisomerase I removes the negative (and positive, in eukaryotictopoisomerase I enzymes) supercoils from DNA. This is an extremelyimportant reaction that allows RNA transcription and DNA replication totake place.

Topo I enzymes are monomeric and transiently break one strand of duplexDNA, allowing for single step changes in the linking number of circularDNAs (the number of times on strand of DNA crosses the other). Topo Ican be divided into two subfamilies: Type IA and Type IB.

Type IA enzymes require magnesium and a single-stranded segment of DNA;

additionally they form a covalent intermediate with the 5′ end of thebroken DNA strand and relax only negatively supercoiled DNA. Type IBtopoisomerase I enzymes do not require any metal cofactors, work ondouble stranded DNA as well, form a covalent intermediate with the 3′end of the broken strand, and are able to relax both positive andnegative supercoils.

Topo I must also seal the break in the DNA. This reversible breakage isachieved because Topo I maintains the high energy status of thephosphodiester bond during unwinding. Topo I uses an enzyme residue(typically a tyrosine) to break DNA. A new enzyme-DNA phosphodiesterbond is formed in the process. The covalent DNA-enzyme intermediate canbe readily attacked by the free end of the DNA because the enzyme-DNAphosphodiester bond is of comparable energy to the orginal bond.

Sequences for Topoisomerase I are known to the art. Examples ofsequences for known Topo I enzymes may be found in the followingpapers/gene databases:

(a) Human Topo I

-   D'Arpa et al. (1988) Proc Natl Acad Sci U S A 85(8):2543-7; NCBI    pubmed nucleotide LOCUS HUMTOPI, ACCESSION J03250    (b) Yeast Topo I-   Thrash et al (1985) Proc Natl Acad Sci U S A 82(13):4374-8; NCBI    pubmed nucleotide LOCUS YSCTOPI, ACCESSION K03077    (c) E.coli Topo I-   Tse-Dinh et al. (1986) J Mol Biol 191(3):321-31; NCBI pubmed    nucleotide LOCUS ECTOPA, ACCESSION X04475 X12873

Human Topo I is of considerable biomedical importance because it is themain target of camptothecin (CPT) family of anticancer drugs. Thesedrugs act by prolonging the lifetime of the nicked intermediate in theTopo I reaction which are presumed to form obstacles to the advancementof transcription and replication complexes that eventually lead to DNAdamage and cell death.

Heat Shock Protein 90 (HSP90) consists of a highly conserved, 25 kDaN-terminal domain connected to a highly conserved, 55 kDa C-terminalregion by a ‘charged linker’, which is variable in both length andcomposition among species and isoforms. The eukaryotic HSP90s areessential and ubiquitous molecular chaperones with key roles in thefolding, activation and assembly of a range of client proteins typicallyinvolved in signal transduction, cell cycle control or transcriptionalregulation.

Sequences for HSP90 are also known to the art. Examples of known HSP90proteins may be found in the following papers/gene databases:

-   (a) Human HSP90 beta: Rebbe et al. Gene 1987;53(2-3):235-45;    GENBANK/M16660; and NCBI PubMed nucleotide LOCUS HUMHSP90-   (B) A bacterial homologue of HSP90 from E.coli (HtpG): Nemoto et al.    Eur J Biochem. 2001 October;268(20):5258-69; swissprot: locus    HTPG_ECOLI, accession P10413 (protein accession number); and NCBI    PubMed protein LOCUS HTPG_ECOLI

Heat Shock proteins exert their effect under conditions of stress suchas heat shock, oxidative, chemical and other stress situations. Thebiochemical function of HSP90 is catalysing the correct folding andmaturation of a number of protein substrates. Without the function ofHSP90 the abnormal conformation of the partner proteins would targetthem for proteolytic degradation.

HSP90 is known to bind to mediators of signalling pathways and otherproteins but it is not known to the art that HSP90 may interact withTopo 1. However the inventors have established that HSP90 and Topo Iinteract.

The inventor has found that the combined use of a first agent thatattenuates Topoisomerase I activity and a second agent that inhibitsHeat Shock Protein 90 activity is highly effective for effectingchemotherapy. The first and second agents may be administeredcontemporaneously (e.g. as a composition according to the third aspectof the invention) or sequentially. If administered sequentially thefirst and second agents should be therapeutically active within thesubject being treated at the same time.

Chemotherapy with first and second agents according to the invention isparticularly useful because such therapy results in surprisingsynergistic actions. The inventors have found that disruption of theinteraction between Topo I and HSP90 causes an increase in DNA damageand thereby kills 3-5 time (or more) the number of proliferating cellsin comparison with what is achievable with a monotherapy. Furthermoresatisfactory therapy may be effected using lower doses than would berequired in a monotherapy. This has the advantage that the toxicside-effects associated with high doses of chemotherapeutic agents maybe obviated or reduced.

For instance, damage to health tissues (and other associated sideeffects of high dose chemotherapy—e.g. sickness, hair loss) may bereduced in human cancer chemotherapy by using lower doses of thecombined agents according to the invention (than would be required in amonotherapy) without comprising the efficacy of the treatment.

The invention is based upon studies that have been orientated towardsthe rational design of chemotherapeutic regimens. The inventor realisedthat drug development up to the present time has only been directedagainst single molecule targets and that rational selection ofcombination chemotherapy may be based on investigating the mechanisms ofaction of chemotherapeutic agents and identifying potential interactionat the cellular targets of such agents. The inventor's studiesestablished that Topo I and HSP90 interact and lead to the realisationthat a combination of agents that specifically inhibit the individualproteins will have great efficacy in chemotherapy. Furtherexperimentation (see the Example) established that treatment of cellswith a combination of agents according to the invention was highlyeffective as a chemotherapy. Furthermore the combination surprisinglyrepresented a synergistic effect. Although the inventor does not wish tobe bound by any hypothesis, it is believed that disrupting theinteraction between the Topo I and HSP90 allows the generation of moreDNA damage, thus killing the dividing cells, than would be possibleusing the agents in monotherapy. We believe the agents have suchefficacy because two targets in a single pathway (the stress responsepathway) are modulated.

Three papers in the prior art contemplate the use of HSP90 inhibitors incombination with other chemotherapeutic agents.

(a) Munster et al. (Clin Cancer Res 2001 August;7(8):2228-36) disclosesthat ansamycin antibiotics such as 17-AAG (an HSP90 inhibitor) andDoxorubicin may be combined in chemotherapy.

(b) Blagosklonny et al. (Leukemia 2001 October;15(10):1537-43) disclosesthat the ansamycin antibiotic (geldanamycin—an Hsp90 inhibitor)sensitises cells to the effects of Taxol or doxrubicin.

(c) Neckers (Trends in Molecular Medcine 2002 Vol(8) s55-s61) disclosesthat HSP90 inhibitors could increase the efficacy of certainchemotherapeutic agents.

However none of the papers contemplate modulation of Topo I. Furthermorethere is no suggestion in any of these papers that agents that modulateTopo I may be combined with HSP 90 inhibitors according to the presentinvention and be used in chemotherapy to result in the sort ofsurprising and synergistic results reported herein.

A skilled person may be motivated to try combination therapies of anHSP90 inhibitor with a wide variety of other chemotherapeutic agents.However most of such combinations would have no beneficial orsynergistic effect and the inventor believes that the efficacy of thespecific combination of first and second agents according to the presentinvention would have been surprising to a skilled person.

Several classes of compound may be used according to the invention asthe first agent. These compounds include:

-   -   (i) compounds that bind to Topo I and inhibit its activity (e.g.        competitive inhibitors; allosteric inhibitors, cleavable complex        inhibitors etc);    -   (ii) compounds which prevent the transcription, translation or        expression of Topo I (e.g. ribozymes or antisense DNA molecules        e.g. antisense crossing the first intron/exon boundary);    -   (iii) compounds which inhibit release of Topo I from        intracellular stores; and    -   (iv) compounds which increase the rate of degradation of Topo I.

Examples of compounds that may be used as first agents are well known tothe art. For instance, Pommier et al. (Biochimica et Biophysica Acta(1998) 1400 p83-106) disclose drugs targeted to Topo I as well asmechanism of action for Topo I. Such drugs are incorporated herein byreference as examples of preferred first agents.

Preferred compounds may attenuate the activity of human Topo I.

The compound may be a Topo I poison or a Topo I suppressor—e.g. asdisclosed in Table 1 Pommier et al. (supra).

A preferred first agent is Gemcitabine(2′,2′-difluoro-2′-deoxycytidine). Gemcitabine is an antimetabolite thatpoisons Topo I (see Pourquier et al. Clin Cancer Res 2002 August 8(8)p2499-2504)

It is preferred that the first agent is Camptothecin (NSC 94600; and CASRegistry Number: 7689034) or a derivative thereof. Examples of preferredderivatives of Camptothecin are disclosed in Pommier et al. (supra) e.g.see FIG. 3 of the paper.

-   -   Camptothecin has the following names and structure:    -   Camptothecin    -   Camptothecine (8CI)    -   CAMPTOTHECIN    -   NSC 100880    -   1H-Pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione,        4-ethyl-4-hydroxy-, (S)-(9CI)    -   20(S)-Camptothecine    -   21,22-Secocamptothecin-21-oic acid lactone

Another preferred first agent is Topotecan (NSC 609699) or a derivativethereof. Topotecan has the following names and structure:

Topotecan

9-Dimethylaminomethyl-10-hydroxycamptothecin, HCl salt

1H-Pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione,

4-ethyl-4,9-dihydroxy-10-[(dimethylamino)methyl]-, HCl salt (S)

Hycamptamine

Another preferred first agent is Irinotecan or a derivative thereof.Irinotecan has the following names and structure:Irinotecan

A further preferred first agent is Camptosar (CPT-11_or a derivativethereof.Camptosar (CPT-11)

Several classes of compound may be used according to the invention asthe second agent. These compounds include:

-   -   (i) compounds that bind to HSP90 and inhibit its activity (e.g.        competitive inhibitors or allosteric inhibitors);    -   (ii) compounds which prevent the transcription, translation or        expression of HSP90 (e.g. ribozymes or antisense DNA molecules);    -   (iii) compounds which inhibit release of HSP90 from        intracellular stores; and    -   (iv) compounds which increase the rate of degradation of HSP90.

Geldanamycin and its derivatives (e.g. 17-Allylamino,17-demethoxygeldanamycin-17-AAG or Macbecin II) are preferred secondagents for use according to the present invention.

Macbecin II

-   Geldanamycin,    18,21-didehydro-6,17-didemethoxy-18,21-dideoxo-18,21-dihydroxy-15-methoxy-6-methyl-11-O-methyl-,    (6S,15R)-(9CI)-   Geldanamycin,    18,21-didehydro-6,17-didemethoxy-18,21-dideoxo-18,21-dihydroxy-15-methoxy-6-methyl-11-O-methyl--   Geldanamycin,    18,21-didehydro-6,17-didemethoxy-18,21-dideoxo-18,21-dihydroxy-15-methoxy-6-methyl-11-o-methyl-,    (6S,1 5R)-    Macbecin II    2-Azabicyclo[16.3.1]docosane, geldanamycin deriv. (9CI)    NSC 330500    CAS Registry Number: 73341738    NSC 122750

Radicicol may be used as a second agent according to the invention.

CNF-101 is a semi-synthetic derivative of Geldanamycin from ConformaTherapeutics (see www.conformacorp.com) and is a further preferredsecond agent.

It will be appreciated that agents may be developed that have a dualaction in that they are able to attenuate Topo I activity and alsoinhibit Hsp90. Such agents may be used in an adaption of the presentinvention which involves the use of a single, dual action, agent onlyrather than separate first and second agents.

The first and second agents may be further combined with othertherapeutics when there is a medical need. For instance, for certainmedical conditions, the inventor has found even greater therapeuticefficacy when the agents are combined with a mediacment which suppressesapoptosis in non-cancerous tissue (eg pifithrin-α).

Agents which attenuate Topo I activity and inhibit Hsp90 may be used inchemotherapy to treat a number of conditions requiring the induction oftargeted cell death. These include:

1) Cancer chemotherapy;

2) antibacterial treatments;

3) antifungal treatments;

4) the treatment of AIDS/HIV;

5) the treatment of multiple sclerosis; and

6) the killing and inhibition of proliferation of any organism.

When used to treat cancer, the agents are particularly effective fortreating solid tumours such as bowel cancer, small cell and non-smallcell lung cancer, head and neck cancer, breast cancer, bladder cancerand malignant melanoma.

The combined agents are also particularly effective for the treatment ofpaediatric tumours such as neuroblastoma and in the treatment ofleukaemias and lymphomas, in which both proteins are contemporaneouslyor sequentially targeted.

The inventors have found that the combination of first and second agentsimproves the effectiveness of the agents for all known clinicalapplications for the agents.

When the agents are used to treat non-mammalian organisms, or to attackmicro-organisms, it is preferred that the agents are effective forattenuating the activity of the species equivalent of Topo I orinhibiting the species equivalent of HSP90. For instance, when theagents are used as antibacterial agents it is preferred that theyattenuate the activity of Topo 1A or an equivalent thereof (see Tse-Dinhet al. supra for E. coli TopA) and inhibit pHtpG (a bacterial equivalentof HSP 90).

We have found that the agents are particularly useful for arresting thegrowth or directly killing a number of bacteria. These include gram −veand gram +ve bacterium.

The agents may be used to treat a number of bacterial infections inmammals (and particularly humans). Bacteria that may be attackedaccording to the invention are listed below. The conditions caused bysuch bacteria, and thereby treatable by the combination therapyaccording to the invention, are indicated in parentheses.

The following bacteria may be treated according to the invention:

Abiotrorphia (Reported infections—endophthalmitis, brain abscess,osteomyelitis);

Achromobacter (Reported infections—septicaemia, CAPD peritonitis,pneumonia, ear infection);

Acidaminococcus (Reported infections—abscesses, post surgicalinfections);

Acidovorax (Reported infections—wound infection, UTI, bacteraemia,meningitis, septic arthritis);

Acinetobacter (Reported infections—septicaemia, UTI, wound infectionsabscesses, endocarditis, meningitis, osteomyelitis);

Actinobacillus (Reported infections—periodontitis, endocarditis,abscesses, pericarditis, meningitis, septicaemia, pneumonia, empyema,hepatitis);

Actinobaculum (Reported infections—pyelonephritis);

Actinomadura (Reported infections—actinomycetoma, madura foot);

Actinomyces (Reported infections—actinomycosis);

Aerococcus (Reported infections—endocarditis, UTI, wound infection,meningitis, abscesses);

Aeromonas (Reported infections—wound infection, abscesses, septicaemia,acute diarrhoea, meningitis, leech bite infection, alligator biteinfection, infections associated with aquatic exposure);

Afipia (Reported infections—cat scratch disease (A. felis), septicarthritis, bone marrow infection (A. broomeae), bone infection (A.clevelandensis);

Agrobacterium (Reported infections—endocarditis, CAPD peritonitis, UTI,line sepsis);

Alcaligenes (Reported infections—pneumonia, otitis, UTI, osteomyelitis,bacteraemia);

Alloiococcus (Reported infections—otitis media);

Amycolata (Please see Pseudonocardia);

Amycolatopsis (Species associated with infection—A. orientalis);

Anaerobospirillum (Reported infections—diarrhoea, bacteraemia);

Anaerorhabdus (Reported infections—lung abscess, appendix absecess,abdominal abscess);

“Anguillina” (Species associated with infection—“Anguillina coli”);

Arachnia (Species associated with infection—Arachnia propionica);

Arcanobacterium (Reported infections—septic arthritis (A. bernardiae andA. pyogenes), UTI and septicaemia (A. bernardiae), tonsillitis,cellulitis, lymphadenitis, brain abscess, septicaemia, osteomyelitis (A.haemolyticum);

Arcobacter (Reported infections—enteric infection (diarrhoea andabdominal cramps);

Arthrobacter (Reported infections—UTI, bacteraemia, Whipple's disease);

Atopobium (Reported infections—UTI, dental abscess, pelvic abscesses,wound infection);

Aureobacterium (With the exception of A. resistens, Aureobacterium spp.have been reclassified as members of the genus Microbacterium. The nameA. resistens (which is vancomycin-resistent) was validly after otherAureobacterium spp. were reclassified as Microbacterium spp.Aureobacterium isolates have been misidentified as “Corynebacteriumaquaticum”);

Bacillus (B. anthracis—the agent of anthrax B. thuringiensis, abiological insecticide has caused corneal infection);

Bacteroides (Reported infections—abscesses, bacteraemia, biteinfections, wound infections, chronic otitis media, pelvic inflammatorydisease);

Balneatrix (Reported infections—pneumonia, bacteraemia, meningitis);

Bartonella (Reported infections—Oroya fever and verruga peruana (B.bacilliformis), cat scratch disease (B. henselae), bacillaryangiomatosis (B. henselae, B. quintana), trench fever (B. quintana),endocarditis (B. elizebethae), bacteraemia (B. vinsonii arupensis);

Bergeyella (Reported infections—wound infection, septicaemia,meningitis);

Bifidobacterium (Reported infections—bacteraemia, peritonitis,abscesses, otitis, paronychia);

Bilophila (Reported infections—appendicitis, abscesses, bacteraemia,biliary tract sepsis);

Branhamella (Species associated with infection—B. catarrhalis, thisorganism has been reclassified as Moraxella catarrhalis);

Borrelia (Species associated with relapsing fever, Reportedinfections—wound infection, septicaemia, meningitis);

Bordetella (Reported infections—respiratory tract infection (B.bronchiseptica, B. paralpertussis, B. pertussis), whooping cough (B.parapertussis, B. pertussis), bacteraemia, otitis, wound infection (B.hinzii, B. holmseii, B. trematum),

Brachyspira (Reported infections—intestinal spirochaetosis);

Brevibacillus (Reported infections—endophthalmitis, food poisoning,bacteraemia);

Brevibacterium (Reported infections—bacteraemia, meningitis, chestinfection);

Brevundimonas (Reported infections—septicaemia);

Brucella (Reported infections—brucellosis);

Burkholderia (associated infections include lung infection, bacteraemia,endocarditis, septic arthritis, UTI, cystic fibrosis patients);

Buttiauxella (Reported infections—appendicitis, wound infection);

Butvrivibrio (Reported infections—endophthalmitis);

Calymmatobacterium (This taxon has been reclassified as Klebsiellagranulomatis);

Campylobacter (associated with diarrhoea, bacteraemia, periodontitis,appendicitis, peritonitis and head and neck infections fever,meningoencephalitis, endocarditis, abscesses and abscess—zoonoses frommammals and birds,

Campylobacter butzleri was reclassified as Arcobacter butzleri C.cinaedi, C. fennelliae, C. pyloridis were reclassified as Helicobacterspp);

Capnocytophaga (wound infection, septicaemia, abscesses, meningitis,endocarditis—associated with dog bites systemic infections inneutropenic patients);

Cardiobacterium (Reported infections—endocarditis, meningitis);

Catonella (Reported infections—periodontitis);

Cedecea (Reported infections—bacteraemia);

Cellulomonas (reported cases of bacteraemia, meningitis cases ofbacteraemia, endocarditis);

Centipeda (Reported infections—periodontitis);

Chlamydia (Reported infections—trachoma, genital infection, neonatalinfection, lymphogranuloma venereum);

Chlamydophila (associated with abortion following contact with infectedruminants, associated with chest infection agent of psittacosis, azoonosis from birds);

Chromobacterium (Reported infections—septicaemia, osteomyelitis,abscesses, eye infection);

Chyseobacterium (Reported infections—bacteraemia, meningitis, abdominalsepsis, wound infection, line infection);

Chryseomonas (Chryseomonas luteola has been reclassified as Pseudomonasluteola);

Citrobacter (Reported infections—UTI, meningitis, haemolytic-uraemicsyndrome);

Clostridium (associated with wound infection, bacteraemia and abscesses,botulism, diarrhoea (usually antibiotic-associated) and pseudomembranouscolitis, food poisoning, necrotising enterocolitis (pigbel, Darmbrand),gas gangrene—(C. histolyticum C. novyi, C. septicum, C. sordellii alsoassociated with gas gangrene), tetanus);

Collinsella (Species associated with infection—Collinsella aerofaciens),

Comamonas (Reported infections—bacteraemia, conjunctivitis);

Corynebacterium (associated with infections such as septicaemia,peritonitis, eye infection, wound infection, endocarditis,osteomyelitis, septic arthritis, meningitis and abscesses diphtheria andcutaneous infection, tropical ulcer, septicaemia, pulmonary infection,lymphadenitis pharyngitis or diphtheria-like illness);

Coxiella (The agent of Q fever);

Cryotobacterium (Associated with periodontitis);

Delftia (Reported cases of bacteraemia and endocarditis);

Dermabacter (brain abscess, bacteraemia, wound infection);

Dermatophilus (Reported to cause cutaneous infection—zoonosis fromcattle, sheep, goats and horses);

Desulfomonas (Associated with pilonidal abscess and peritonitis);

Desulfovibrio (Reported infections—bacteraemia, liver abscess);

Dialister (Reported infections—periodontitis);

Dichelobacter (Reported infections—pilonidal cyst, rectal fistula, woundinfection);

Dolosicoccus (Reported infections—bacteraemia);

Dolosigranulum (Reported infections—spinal cord infection, eyeinfection);

Edwardsiella (Reported infections—wound infections, abscesses,gastroenteritis—associated with aquatic exposure and penetrating fishinjury);

Eggerthella (Reported infections—rectal abscess);

Ehrlichia (Reported infections—Ehrlichiosis);

Eikenella (Reported infections—septicaemia, endocarditis, abscesses,septic arthritis);

Empedobacter (Species associated with infection—E. brevis);

Enterobacter (Associated infections—bacteraemia, respiratory tractinfections, UTI—associated with nosocomial infection);

Enterococcus (Associated infections—bacteraemia, abscesses,endocarditis, meningitis, UTI, peritonitis, osteomyelitis, woundinfection);

Erwinia (Associated infections—UTI);

Erysipelothrix (Associated infections—erysipeloid, septicaemia,endocarditis);

Escherichia (associated with UTI, bacteraemia, wound infection,meningitis, enteric infection, haemolytic uraemic syndrome);

Eubacterium (Associated infections—wound infection, abscesses,septicaemia, periodontitis);

Ewingella (Associated infections—septicaemia, wound infection, UTI);

Exiguobacterium (Species associated with infection—E. acetyliticum, E.aurantiacum),

Facklamia (Associated infections—UTI, bacteraemia, abscess);

Filifactor (Associated infections—gingivitis, periodontitis);

Flavobacterium (Associated infections—bacteraemia, diarrhoea);

“Flexispira” (Associated infections—bacteraemia, diarrhoea);

Francisella (associated with septicaemia and invaxsive systemicinfection, tularaemia);

Fusobacterium (Associated infections—abscesses, bacteraemia,periodontitis, endocarditis, necrobacillosis);

Gardnerella (Associated infections—intrauterine and neonatalsepsis—associated with bacterial vaginosis);

Gemella (Associated infections—bacteraemia, endocarditis);

Globicatella (Associated infections—bacteraemai, UTI, meningitis);

Gordona (Associated infections—pulmonary infection, sternal woundsepsis, brain abscess, bacteraemia);

Haemophilus (associated with Brazilian purpuric fever; associated withsinusitis, otitis media, pneumonia, abscesses, endocarditis; the agentof chancroid; associated with bacteraemia, meningitis, epiglottitis,respiratory tract infection);

Hafnia (Associated infections—bacteraemia—has been associated with casesof diarrhoea);

Helicobacter (a zoonosis from dogs and hamsters cause ofgastroenteritis; associated with septiciaemia and proctitis; sepicaemiain a neonate; gastritis);

Helococcus (associated with sebaceous cyst infection and breastabsecess);

Holdemania (Species associated with infection—H. filiformis);

Ignavigranum (Associated infections—wound infection, ear abscess);

Johnsonella (Associated infections—perodontitis);

Kingella (Associated infections—septic arthritis, endocarditis);

Klebsiella (associated with UTI, bacteraemia, wound infection,respiratory tract infection; rhinoscleroma);

Kocuria (Species associated with infection—K. varians, K. kristinae);

Koserella (Associated infections—wound infection, septic arthritis);

Kurthia (bacteraemia and endocarditis; diarrhoea);

Kytococcus (Species associated with infection—K. sedentarius);

Lactobacillus (Associated infections—abscesses, bacteraemia,endometritis, endocarditis, lung infection, UTI—reported risk factorsfor infection, surgery, malignacy, diabetes mellitus, immunodeficiency);

Lactococcus (Associated infections—bacteraemia, endocarditis, UTI);

Lautropia (has been isolated from oral flora of an HIV patient and fromsputum of a cystic fibrosis patient);

Leclercia (Associated infections—bacteraemia, wound infection);

Legionella (Associated infections—legionaires' disease, Pontiac fever);

Leminorella (Associated infections—UTI);

Leptospira (Associated infections—leptospirosis);

Leptotrichia (Associated infections—bacteraemia, endocarditis);

Leuconostoc (Associated infections—meningitis, bacteraemia, pulmonaryinfection);

Listeria (Associated infections—septicaemia, meningitis, intra-uterineinfection, enteric infection);

Megasphaera (Associated infections—septicaemia, meningitis,intra-uterine infection, enteric infection);

Methylobacterium (Associated infections—bacteraemia, CAPD peritonitis);

Microbacterium (Associated infections—endophthalmitis, UTI,endocarditis, soft tissue infection, hypersensitivity pneumonitis,meningitis, CAPD peritonitis);

Micrococcus (Associated infections—bacteraemia, endocarditis, septicarthritis);

Mitsuokella (Species associated with infection—M. multiacida);

Mobiluncus (Associated infections—endometritis,chorioamnionitis—associated with bacterial vaginosis);

Moellerella (diarrhoea);

Moraxella (associated with—conjunctivitis, wound infection,endocarditis, abscesses, osteomyelitis);

Morganella (Associated infections—bacteraemia, UTI, wound infection);

Mycobacterium (Leprosy, cervical adenitis, Buruli ulcer, fish-tankgranuloma, M. malmoense, M. szulgai M. kansasii, M. xenopi—associatedwith pulmonary infection, systemic infection in imunocompromisedpatients, post-inoculation infection);

Mycoolasma (Associated infections—respiratory infection, post-partumfever, pyelonephritis, pelvic inflammatory disease, myocarditis,pericarditis, meningitis);

Myroides (Associated infections—UTI, wound infection);

Neisseria (associated with meningitis, bacteraemia, endocarditis,osteomyelitis, agent of genital gonorrhoea, septicaemia, ophthalmianeonatorum, associated with septicaemia, meningitis, conjunctivitis,genital infection, epiglottitis)

Nocardia (nocardiosis);

Nocardiopsis (Associated infections—mycetoma, cutaneous infection,pulmonary infection, conjunctivitis);

Ochrobactrum (Associated infections—bacteraemia, endophthalmitis, liverabscess—reported association with nosocomial infections in debilitatedpatients);

Oeskovia (associated with meningitis, pyelonephrosis, CAPD peritonitis,endophthalmitis);

Oligella (associated with UTI, septicaemia—infection associated withurinary catheters);

Orientia (Associated infections—scrub typhus);

Paenibacillus (Associated infections—septicaemia, meningitis,pneumonia);

Pantoea (Associated infections—bacteraemia, endocarditis, woundinfection, cellulitis, alligator bite infection, endophthalmitis);

Parachlamydia (Parachlamydia acanthamoebae has been associated withhypersensitivity pneumonitis (humidifier fever);

Pasteurella (Associated infections—wound infection, septicaemia,abscesses, pneumonia, endocarditis, meningitis—infections relate tospp);

Pediococcus (Associated infections—bacteraemia, abscesses, pulmonaryinfection—infections in debilitated patients);

Peptococcus (Peptococcus niger has been associated with anaerobicinfections including intra-abdominal sepsis);

Photobacterium (Associated with necrotising wound infection);

Photorhabdus (Associated infections—bacteraemia, wound infection);

Plesiomonas (Associated infections—gastroenteritis, septicaemia,meningitis, endophthalmitis);

Porphyrimonas (Associated infections—mixed anaerobic infections atvarious sites, periodontitis, associated with bite infections (human andanimal);

Prevotella (Associated infections—abscesses, bacteraemia, woundinfection, bite infections, genital tract infections, periodontitis);

Propionibacterium (Associated infections—abscesses, endocarditis,bacteraemia, spetic arthritis, endophthalmitis, acne vulgaris);

Proteus (Associated infections—UTI, bacteraemia, wound infection,abscesses);

Providencia (Associated infections—UTI, wound infection, bacteraemia);

Pseudomonas (Reported infections—bacteraemia, UTI, wound infection,abscesses, septic arthritis, conjunctivitis, endocarditis, meningitis,CAPD peritonitis—nosocomial)

Pseudonocardia (Species associated with infection—P. autotrophica)

Pseudoramibacter (Associated infections—periodontal disease, woundinfection, abscesses)

Psychrobacter (Associated infections—meningitis, bacteraemia, eyeinfection);

Rahnella (Associated infections—UTI, septicaemia);

Ralstonia (Associated infections—bacteraemia, UTI, meningitis, woundinfection, peritonitis);

Rhodococcus (associated with bacteraemia, osteomyelitis, lungabscesses—infections of immunocompromised patients including AIDS);

Rickettsia (Associated infections—rickettsial spotted fever, ticktyphus, tick bite fever, rickettsialpox);

Roseomonas (Associated infections—bacteraemia, wound infection,peritonitis);

Rothia (Associated infections—endocarditis, abscesses);

Ruminococcus (Associated infections—abdominal sepsis, abscesses);

Salmonella (Associated infections—gastroenteritis, enteric fever,osteomyelitis);

Selenomonas (Associated infections—bacteraemia, lung abscess—infectionsreported to be associated with malignancy or alcohol abuse);

Serratia (Associated infections—septicaemia, abscesses, bum infections,osteomyelitis);

Shewenella (associated with cases of intra-abdominal sepsis, meningitisand bacteraemia);

Shigella (Associated infections—enteric infection);

Simkania (Associated infections—bronchiolitis, pneumonia);

Slackia (Associated infections—periodontitis);

Sphingobacterium (Associated infections—bacteraemia, UTI, peritonitis);

Sphingomonas (Associated infections—septicaemia, UTI, wound infections,CAPD peritonitis—nosocomial infections);

Spirillum (Associated infections—rat bite fever);

Staphylococcus (Associated infections—Bacteraemia, wound infection,endocarditis, catheter-related sepsis,UTI, toxic shock syndrome, eyeinfection, osteomyelitis);

Stenotrophomonas (associated with various (mostly nosocomial)infections—bacteraemia, meningitis, wound infection, UTI and pneumonia);

Stomatococcus (Associated infections—endocarditis, meningitis,neutropenic sepsis);

Streptobacillus (Associated infections—rat bite fever, Haverhill fever);

Streptococcus (Associated infections—pharyngitis, bacteraemia, pyogenicinfection, necrotising infection, septic arthritis, glomerulonephritis,meningitis, rheumatic fever, abscesses, endocarditis, pharyngitis, woundinfection, pneumonia, pericarditis, CAPD, peritonitis, sinusitis,otitis, conjunctivitis);

Streptomyces (Associated infections—actinomycetoma);

Succinivibrio (Associated infections—bacteraemia);

Sutterella (Associated infections—appendicitis, peritonitis, abascesses,osteomyelitis);

Suttonella (Associated infections—endocarditis, eye infection);

Tatumella (Associated infections—bacteraemia, UTI);

Tissierella (Associated infections—bacteraemia);

Trabulsiella (Associated infections—diarrhoea);

Treponema (associated with periodontal disease, pinta, genital lesions,venereal and non-venereal endemic syphilis, yaws);

Tropheryma (associated with Whipple's disease);

Turicella (Associated infections—otitis, cervical abscess);

Ureaplasma (Associated infections—urethritis);

Vagococcus (Species associated with infection—V. fluvialis);

Veillonella (Associated infections—abscesses, bacteraemia);

Vibrio (The agent of cholera, associated with wound infection,bacteraemia, diarrhoea and septiciaemia, septicaemia, meningitis,endometritis);

Weeksella (associated with peritonitis and vaginal infections);

Xanthomonas (bacteraemia);

Yersinia (agent of plague, associated infections—enterocolitis, softtissue infections, mesenteric lymphadenitis, enteric infection); and

Yokenella (Associated infections—bacteraemia, wound infection).

The combination therapy may also be used to treat fungal infections of asubject. The agents are effective against the following fungi: Candidaspp, Aspergillus spp. Malassezia spp, Trichosporon spp, Fusarium spp,Paecilomyces spp and Acremonium spp, also Rhizopus, Mucor, Absidia,Blastomyces spp, Coccidiodes spp, Cryptococcus spp, Histoplasma spp

The inventor has also found that the combination therapy may also beused to treat a number of parasitic infections of a subject. Forinstance, the combination therapy is useful for treating malaria.

The inventors have found that the combination therapy is particularlyuseful for treating infection (e.g. local or systemic or deep systemicinfections) associated with immune suppressed patients; urinary tract,bloodstream infections and pneumonia.

The agents may be used to treat existing medical conditions but may alsobe used when prophylactic treatment is considered medically necessary.

The agents used according to the invention may take a number ofdifferent forms depending, in particular on the manner in which they areto be used. Thus, for example, the agents may be in the form of apowder, tablet, capsule, liquid, ointment, cream, gel, hydrogel,aerosol, spray, micelle, liposome or any other suitable form that may beadministered to a person or animal. It will be appreciated that thevehicle for the agents should be one which is well tolerated by thesubject to whom it is given and enables delivery of the agent to thetarget tissue.

The agents may be used in a number of ways. For instance, systemicadministration may be required in which case the agents may be containedwithin a composition, which may, for example, be ingested orally in theform of a tablet, capsule or liquid. Alternatively the agents may beadministered by injection into the blood stream. Injections may beintravenous (bolus or infusion) or subcutaneous (bolus or infusion). Thecompounds may also be administered by inhalation (e.g. intranasally).

The agents may also be incorporated within a slow or delayed releasedevice. Such devices may, for example, be inserted under the skin andthe compound may be released over weeks or even months. The devices maybe particularly advantageous when an agent is used which would normallyrequire frequent administration (e.g. at least daily ingestion of atablet or daily injection).

It is preferred that second agents according to the invention areinitially dissolved in solvents such as DMSO before dilution in aqueoussolution for the preparation of liquid medicaments.

The agents may be formulated as prodrugs. Such prodrugs may be stored asinactive and stable medicaments which are subsequently activated.

It will be appreciated that the amount of an agent required isdetermined by biological activity and bioavailability that in turndepends on the mode of administration and the physicochemical propertiesof the agents employed. The frequency of administration will also beinfluenced by the abovementioned factors and particularly the half-lifeof the agents within the subject being treated.

Known procedures, such as those conventionally employed by thepharmaceutical industry (e.g. in vivo experimentation, clinical trialsetc), may be used to establish specific formulations of agents andprecise therapeutic regimes (such as daily doses and the frequency ofadministration).

Generally, a daily dose of between 0.01 μg/kg of body weight and 1.0g/kg of body weight of a first agent and a second agent may be used forchemotherapy depending upon which specific agents are used. Morepreferably the daily dose of each agent is between 0.1 μg/kg of bodyweight and 100 mg/kg of body weight.

Generally, a daily dose of 1 ng-1 g/M² (per agent) of both a first agentand a second agent may be used for chemotherapy in humans—depending uponwhich specific agents are used.

Purely by way of example suitable doses of first agents (e.g.Irinotecan, Topotecan, Camptothecin, Gemcitabine and derivatives andanalogues thereof) according to the invention for treating a humancancer is 1 ng-1 g/M² IV (depending upon the health status of theindividual) whereas 1 μg-1 g/kg is a suitable does for use in animals.

Purely by way of example suitable doses of second agents doses (forcancer chemotherapy or the treatment of micro organisms) according tothe invention are:

-   (a) A suitable dose of Radicicol (or a derivative or analogue    thereof) for treating a human cancer is 1 ng-1 gM² (depending upon    the health status of the individual).-   (b) A suitable dose of Geldanamycin for treating a human cancer is 1    ng-1 g/M².-   (c) A suitable dose of 17-AAG for treating a human cancer is 1 ng-1    g/M².

For all agents it is preferred that about 1 μg-1 g/kg of a first or asecond agent is used for vetinary purposes. For instance about 4-25mg/kg of Geldanamycin may be used.

Daily doses may be given as a single administration (e.g. a daily tabletfor oral consumption or as a single daily injection). Alternatively theagents used may require administration twice or more times during a day.A patient receiving treatment may take a first dose upon waking and thena second dose in the evening (if on a two dose regime) or at 3 or 4hourly intervals thereafter. Alternatively a slow release device may beused to provide optimal doses to a patient without the need toadminister repeated doses. A preferred route of administration is byintravenous infusion. Administration may be over several hours or evendays.

A preferred means of using protein or peptide agents is to deliver suchagents to the target tissue by means of gene therapy. For instance, genetherapy may be used to decrease expression of Topo I or HSP90, decreaseexpression of enzyme(s) responsible for the intracellular synthesis ofTopo I or HSP90, increase expression of a protein which promotesbreakdown of Topo I or HSP90. Therefore according to a fourth aspect ofthe present invention there is provided a delivery system for use in agene therapy technique, said delivery system comprising:

-   -   (i) a first DNA molecule encoding for a protein which directly        or indirectly attenuates Topoisomerase I activity; and    -   (ii) a second DNA molecule encoding for a protein which directly        or indirectly inhibits Heat Shock Protein 90 activity;        wherein said DNA molecules are capable of being transcribed to        allow the expression of said proteins and thereby be effective        for chemotherapy.

The delivery systems according to the fourth aspect of the invention arehighly suitable for achieving sustained levels of a protein which arechemotherapeutically active over a longer period of time than ispossible for most conventional therapeutic regimes. The delivery systemmay be used to induce continuous protein expression from cells in atarget tissue that have been transformed with the DNA molecule.Therefore, even if the proteins have a very short half-life as agents invivo, therapeutically effective amounts of the proteins may becontinuously expressed from the treated tissue.

Furthermore, the delivery system of the invention may be used to providethe DNA molecules (and thereby the proteins which are active therapeuticagents) without the need to use conventional pharmaceutical vehiclessuch as those required in tablets, capsules or liquids.

The delivery system of the present invention is such that the DNAmolecules are capable of being expressed (when the delivery system isadministered to a patient) to produce proteins that directly orindirectly have activity for attenuating Topo I activity and inhibitingHSP90 activity. By “directly” we mean that the product of geneexpression per se has the required activity. By “indirectly” we meanthat the product of gene expression undergoes or mediates (e.g. as anenzyme) at least one further reaction to provide an agent effective forattenuating Topo I activity or inhibiting HSP90 activity.

The DNA molecules may be contained within a suitable vector to form arecombinant vector. The vector may for example be a plasmid, cosmid,virus or phage.

Such recombinant vectors are highly useful in the delivery systems ofthe invention for transforming cells with the DNA molecule.

The recombinant vector may also further comprise a promoter or regulatorto control expression of the gene as required.

It will be appreciated that the first and second DNA molecules may becontained within a single vector and the expression thereof may bedriven from either a single promoter or individual promoters.Alternatively the delivery system may comprise first and second DNAmolecules contained within respective first and second expressionvectors.

Recombinant vectors may also include other functional elements. Forinstance, recombinant vectors can be designed such that the vector willautonomously replicate in the cell. In this case, elements that induceDNA replication may be required in the recombinant vector. Alternativelythe recombinant vector may be designed such that the vector andrecombinant DNA molecule integrates into the genome of a cell. In thiscase DNA sequences which favour targeted integration (e.g. by homologousrecombination) are desirable. Recombinant vectors may also have DNAcoding for genes that may be used as selectable markers in the cloningprocess.

The DNA molecules may (but not necessarily) be one which becomesincorporated in the DNA of cells of the subject being treated.Undifferentiated cells may be stably transformed leading to theproduction of genetically modified daughter cells (in which caseregulation of expression in the subject may be required e.g. withspecific transcription factors or gene activators). Alternatively, thedelivery system may be designed to favour unstable or transienttransformation of differentiated cells in the subject being treated.When this is the case, regulation of expression may be less importantbecause expression of the DNA molecules will stop when the transformedcells die or stop expressing the proteins (ideally when chemotherapy isno longer required).

The delivery system may provide the DNA molecules to the subject withoutthem being incorporated in a vector. For instance, the DNA molecules maybe incorporated within liposomes or virus particles. Alternatively the“naked” DNA molecules may be inserted into a subject's cells by asuitable means e.g. direct endocytotic uptake.

The DNA molecules may be transferred to the cells of a subject to betreated by transfection, infection, microinjection, cell fusion,protoplast fusion or ballistic bombardment. For example, transfer may beby ballistic transfection with coated gold particles, liposomescontaining the DNA molecules, viral vectors (e.g. adenovirus) and meansof providing direct DNA uptake (e.g. endocytosis) by application of theDNA molecules directly to the target tissue topically or by injection.

The discovery that Topo I and HSP90 interact has enabled the inventor todevelop a drug screening assay system for testing the efficacy ofcandidate drugs as chemotherapeutic agents. Therefore the twointeracting proteins HSP90 and Topoisomerase I may be used as a complextarget for new drug development in which both proteins arecontemporaneously or sequentially targeted for new mammalian, fungal andanti bacterial agents.

According to a fifth aspect of the present invention there is provided amethod of screening a first and a second compound, to test whether ornot said compounds has efficacy for use in combination as achemotherapy, comprising:

-   -   (i) exposing said compounds to Topoisomerase I and evaluating        whether or not said compounds bind thereto;    -   (ii) exposing said compounds to Heat Shock Protein 90 and        evaluating whether or not said compounds bind thereto; and    -   (iii) selecting a first and second compound, wherein at least        one compound binds to Topoisomerase I and at least one compound        binds to Heat Shock Protein 90 for use in combination as a        chemotherapy.

It will be appreciated that the method according to the fifth aspect ofthe invention may be adapted such that it is used to test whether or nota single compound may have a novel use in chemotherapy. Thereforeaccording to a sixth aspect of the invention there is provided a methodof screening a compound, to test whether or not said compound hasefficacy for use in chemotherapy, comprising exposing said compound toTopoisomerase I and Heat Shock Protein 90 to evaluate whether or notsaid compound prevents interaction between Topoisomerase I and HeatshockProtein 90.

Compounds screened according to the fifth or sixth aspects of theinvention represent candidate chemotherapeutic agents. The screeningmethods are is based upon the inventors realisation that interactionbetween Topoisomerase I and Heat Shock Protein 90 is closely related toundesirable cell growth (carcinogenesis and the like). It will beappreciated that the pharmaceutical industry will be able to use themethods according to the fifth or sixth aspect of the invention toidentify candidate medicaments for further investigation as anti-canceragents.

A preferred technique for carrying out the methods of the fifth andsixth aspects of the invention is to exposure the compounds to be testedto Topo I and HSP90 used as binding partners in an interaction trap.Many forms of interaction trap are known to the art. Preferably a yeasttwo-hybrid interaction trap is employed. Yeast two-hybrid screening is astrategy for screening for interaction between proteins. Yeasttwo-hybrid screening used according to the invention may involveexpression of translational fusions of (a) Topoisomerase I and part of areporter gene; and (b) Heat Shock Protein 90 fused in-frame with theother part of the reporter gene. When the fusion proteins are expressed,interaction between (a) and (b) allows the reporter to assemble andgenerate a signal. Test compounds that represent candidatechemotherapeutic agents prevent interaction between (a) and (b) and maybe identified because no reporter signal is produced from samplescontaining the candidate.

It will be appreciated that any other form of interaction trap may beused to put the invention into practice. Suitable examples includedtechniques such as mammalian two-hybrid, bacterial two-hybrid oralternatively various types of pull down assay.

When the methods relate to the disruption of protein-proteininteractions based on the yeast two-hybrid technique it is preferredthat yeast are used that are permeable to the tested compounds. Examplesof drug permeable yeast which may be used according to the inventioninclude MDS or ISE 2 mutations (e.g. strains carrying these mutations(ISE2), JJ700, BJ201). Suitable strains are disclosed in Hammonds et al.Antimicrob Agents Chemother. 1998 April;42(4):889-94.

It will be appreciated that the methods according to the fifth or sixthaspects of the invention may be adapted to identify compounds thatpromote interaction between Topoisomerase I and Heat Shock Protein 90(rather than inhibit such interaction) Such an adapted test represents agood method for evaluating whether or not a test compound is likely tobe carcinogenic. Therefore according to a seventh aspect of the presentinvention there is provided a method of screening a compound, to testwhether or not said compound is carcinogenic, comprising exposing saidcompound to Topoisomerase I and Heat Shock Protein 90 to evaluatewhether or not said compound promotes interaction between TopoisomeraseI and Heat Shock Protein 90.

Accordingly any compound, identified according to the seventh aspect ofthe invention, that promotes interaction between Topoisomerase I andHeat Shock Protein 90 is likely to be carcinogenic. The method may beused to screen compounds to assess whether or not they are safe to beused by the public. For instance cosmetics, foodstuffs, candidatetherapeutic agents etc may all be tested to investigate whether or notthey may cause cancer. The method according to the seventh aspect of theinvention may also be used for environmental monitoring. For instance,the test may be used to evaluate whether or not effluent from a factorymay contain carcinogenic compounds.

The discovery that Topo I and HSP90 interact has further enabled theinventor to develop a test whereby the measurement of HSP90 and Topo Iprotein levels in cells is used as a diagnostic aid. According to aneighth aspect of the present invention there is provided an in vitromethod for diagnosing whether or not a subject has, or is likely todevelop cancer, comprising:

-   -   (i) detecting the level of activity or expression levels of        HSP90 and Topoisomerase I from a sample of cells from said        subject; and    -   (ii) comparing the level of activity or expression levels of        HSP90 and Topoisomerase I in said sample relative to activity        expression levels of HSP90 and Topoisomerase I from a        non-cancerous sample.

The method according to the eighth aspect of the invention indicatesthat a subject is at risk of developing cancer if the activity orexpression levels of Topo I or HSP90 are raised relative to controlvalues (e.g samples from an individual without cancer or fromnon-cancerous tissues from the subject).

Preferably a first sample is taken from a tissue which is suspected tobe cancerous and a second sample is taken from normal tissue (i.e.non-cancerous tissue) from the same subject.

The method according to the eighth aspect of the invention may beadapted for determining the sensitivity of a subject to a specificcombination of first and second agents according to the invention (i.e.an HSP90 inhibitor and a Topo I inhibitor). Thus according to a ninthaspect of the present invention there is provided an in vitro method forevaluating the suitability of chemotherapeutic treatment foradministration to a subject, comprising:

-   -   (i) detecting the level of activity or expression levels of        HSP90 and Topo I from a sample of cells from said subject; and    -   (ii) comparing the level of activity or expression levels of        HSP90 and Topo I in said sample relative to activity expression        levels of HSP90 and Topo I from a non-cancerous sample.

According to a tenth aspect of the present invention there is providedan in vitro method for monitoring the effectiveness of a chemotherapyfor treating a subject, comprising:

-   -   (i) detecting the level of activity or expression levels of        HSP90 and Topo I from a sample of cells from said subject; and    -   (ii) comparing the level of activity or expression levels of        HSP90 and Topo I in said sample relative to activity expression        levels of HSP90 and Topo I from a non-cancerous sample.

The invention will be further illustrated with reference to non-limitingExamples and figures, in which:

FIG. 1 illustrates the results of an immuno-precipitation assay with anHSP90 antibody in which protein from HCT116p53wt was subjected to aWestern blot using Topo I as a probe as described in Example 1;

FIG. 2 illustrates the results of an immuno-precipitation assay with aTopo I antibody in which protein from HCT116p53wt was subjected to aWestern blot using HSP90 as a probe as described in Example 1;

FIG. 3 illustrates the results of co-immunoprecipitation (IP) westernblots from HCT116 extracts: (A) Immunoprecipitation with ananti-topoisomerase I antibody and probed with an antibody against Hsp90;as a negative control the blot was also probed with an antibody againstPKCα; a non-reactive antibody was used as a non-specific bindingcontrol; and (B) an IP with anti Hsp90 antibody probed with atopoisomerase I antibody as described in Example 1;

FIG. 4 illustrates the effect of Irinotecan alone on cell growth in (A)HCT116 p53 Wild type (WT) cells and (B) HCT116 p53 knockout cells asdescribed in Example 2;

FIG. 5 illustrates the effect of Geldanamycin alone on cell growth in(A) HCT116 p53 Wild type (WT) cells and (B) HCT116 p53 knockout cells asdescribed in Example 2;

FIG. 6 illustrates the effect of a combined treatment of Irinotecan andGeldanamycin on cell growth (the effect being independent of p53 status)in (A) HCT116 p53 Wild type (WT) cells and (B) HCT116 p53 knockout cellsas described in Example 2;

FIG. 7 illustrates the growth inhibition effect of 1.5 μM Irinotecan+25nM Geldanamycin Combination Treatment on (A) HCT116 p53 WT cells and (B)p53 KO cells as described in Example 2;

FIG. 8 illustrates the inhibitory effect of 0.8 μM IRT and 100 nm RDCombination Treatment on (A) HCT116 p53 WT cells and (B) p53 KO cells asdescribed in Example 2;

FIG. 9 illustrates growth inhibition caused by topotecan/geldanamycincombination treatment on (A) HCT116 p53 WT cells and (B) p53 KO cells asdescribed in Example 2;

FIGS. 10 illustrates the results of a clonogenic assays investigatingthe inhibitory effects of Irinotecan (IRT), Geldanamycin (GA) and thecombination of IRT plus GA on the growth of both, HCT116 WT and HCT16 KOcells (the effect being independent of p53 status): (A) illustratescombination therapy results obtained with 15μM IRT plus 1.25 μM GA; and(B) shows results for 50 μM IRT plus 1.25 μM GA;

FIGS. 11 illustrates the cell killing response of of HCT116 p53 KO cellsafter treatment with (A) geldanamycin, (B) irinotecan and (C)geldanamycin/irinotecan in combination, respectively as described inExample 2;

FIG. 12 shows an isobologram illustrating the cell killing responseafter treatment with irinotecan, geldanamycin andirinotecan/geldanamycin in combination on (A) HCT116 p53 WT cells and(B) p53 KO cells as described in Example 2; and

FIGS. 13 illustrates an interaction between topoisomerase I and HtpG asdemonstrated by the results of co-immunoprecipitation (IP) western blotsof IPs from E.coli extracts wherein: TE=total cell extract, IP=proteincomplex following immunoprecipitation; (A) shows results obtained fromimmunoprecipitation with an anti-topoisomerase I antibody in which thewestern blot was probed with an anti-body against Hsp90 (whichrecognises the bacterial equivalent HtpG); and (B) shows resultsobtained from immunoprecipitation with an anti Hsp90 antibody whichrecognises the bacterial equivalent HtpG whilst the western blot wasprobed with a topoisomerase I antibody.

EXAMPLE 1

Experiments were conducted that established HSP90 and Topoisomerase Iinteract and influence cell growth. This discovery lead the inventor todevelop the various aspects of the invention described herein.

1.1 METHODS

Established Cell Culture

The isogenic p53 human colon cancer cell line (WT and KO), HCT116 (seeHwang et al. Nat Med 2001 November 7(11):1255). Cells were maintained inMcCoys 5A medium (Sigma) supplemented with 10% foetal calf serum (Gibco)at 37° C. in a 5% CO₂ enriched humidified environment, Penicillin andStreptomycin.

Standard cell lines as above except:

K562 RMPI 1640 (Sigma), SK-MEL-3 McCoys (Sigma), OAW42 DMEM supplementedwith 1 mM sodium pyruvate 10 μg/ml insulin & NCI-H125 RPMI 1640 (Sigma),HT29 DMEM (Sigma).

Immunoprecipitations

100 mm dishes were seeded with 3—10⁶ cells and allowed to adhereovernight. Media was placed with fresh media alone (control) orcontaining a Topo I inhibitor (e.g. Irinotecan) for 24 hours. Cells werewashed twice with wash buffer (0.4 mM EDTA, 10 mM sodium fluoride, 10 mMsodium pyrophosphate, 0.4 mM sodium orthovanadate) and incubated on icewith 250 μl cell lysis buffer (50 mM Tris HCl pH 8.0, 425 mM NaCl, 1 mMEDTA, 10 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1% v/v igepalCA-630, 5% w/v deoxycholic acid, 0.1% w/v SDS) containing proteaseinhibitor cocktail III (Calbiochem). Cells were scraped on ice,sonicated for 30 seconds and cell debris removed by centrifugation at14,000×g for 30 minutes at 4° C. Cell lysates were then pre-cleared byincubation with 25 μl of 10% w/v protein A sepharose CL-4B (AmershamPharmacia Biotech) in PBS for 1 hour rotating at 4° C. Samples were spunbriefly at maximum speed in a 4° C. benchtop centrifuge and supernatantsremoved to fresh microfuge tubes. 5 μg of either anti-topoisomerase I oranti-heat shock protein 90β (Labvision) antibodies were added to celllysates and incubated at 4° C. overnight. 50 μl of 10% w/v protein Asepharose in PBS was added and samples allowed to precipitate byrotating at 4° C. for 1 hour.

Samples were spun briefly at maximum speed in a 4° C. benchtopcentrifuge and supernatants discarded. Immunoprecipitates were washedwith 250 μl cell lysis buffer, resuspended in 60 μl IPG buffer (7 Murea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris base, 1% w/v DTT) andanalysed by one-dimensional (1-D) electrophoresis.

1-D Electrophoresis and Immunoblotting

Total protein extracts and immunoprecipitations were separated by 7.5%or 12% SDS-PAGE under reducing conditions. Gels were then either stainedusing Colloidal blue concentrate (Sigma) in 20% v/v methanol or blottedonto nitrocellulose membrane. Blots were probed with either rabbitprimary antibodies against human Topoisomerase I or Heat Shock Protein90β, or mouse primary antibodies against human heat shock protein 70(Labvision). Anti-rabbit and anti-mouse IgG secondary antibodiesconjugated with horseradish peroxidase (DAKO) were detected bySupersignal West Dura Extended Substrate (Pierce) and imaged using aFluor-S bioimager (BioRad).

1.2 Results

Protein-Protein Interactions

Protein association studies were conducted using 1 dimensional SDS-PAGEanalyses of co-immunoprecipitated proteins. Irumunoprecipitation wereundertaken with commercial antibodies against the native protein, andbinding partners were identified by 1 dimensional SDS-PAGE (see FIG. 1).

The counter precipitation was performed (IP with antibodies againstHSP90β) and topoisomerase I was demonstrated to be associated withHSP90. FIG. 2 shows western blots of counter immunoprecipitations andprobing of the blots, demonstrating that the corresponding proteins comedown in pull down experiments.

Interaction between topoisomerase I and Hsp90 was demonstrated by afurther set of immunoprecipitation assays. FIG. 3 A illustrates theresults of immunoprecipitation with an anti-topoisomerase I antibodyfollowed by probing of the western blot with an antibody against Hsp90.In addition, this figure shows the outcome of the negative control inwhich the blot was also probed with an antibody against PKC a, anon-reactive antibody (non-specific binding control). FIG. 3B shows theresult of immunoprecipitation with an anti Hsp90 antibody which wasfollowed by probing of the western blot with a topoisomerase I antibody.Overall, this study demonstrates an interaction between topoisomerase Iand Hsp90.

Drug Target

The inventor realised that the interaction between these two proteinsrepresents a new drug target and went on to assess the effect ofmodulators of these proteins in combination for chemotherapy (seeExample 2)

EXAMPLE 2

Example 1 illustrates there was a physical interaction between Topo Iand HSP90. The inventor therefore tested the effect of combining drugsthat had a specific effect on Topo I and a specific effect on HSP90. Thecombination of an HSP90 inhibitor and a topoisomerase I inhibitor show asynergistic effect (see below).

2.1 Methods

2.1.1 Growth Inhibition Assay

96 well flat-bottomed plates were seeded with 3×10³ cells per well andallowed to adhere overnight. Media was then replaced with fresh mediaalone (control) or containing test drugs e.g. 0-100 μM irinotecan, 50 to200 nM geldanamycin (GA) and combinations of both. At fixed time points,cells were fixed with 3:1 methanol:acetic acid and stained with 0.4% w/vsulforhodamine B (Sigma) in 1% v/v acetic acid for 30 minutes. Plateswere then washed twice with 1% v/v acetic acid, the dye solubilised with100 μl per well of 10 mM Tris pH 10.4 and read at A570nm using aBenchmark microplate reader (BioRad).

2.1.2 Clonogenic Assay

Cells were plated at a density of 1000 cells per well in 6 well platesand allowed to adhere overnight. Cells were treated with e.g. 0.5 to 50μM VP16, 50 to 1500 nM GA or combinations of the two for 1 hour. Cellswere then washed twice with PBS and re-incubated with fresh medium for10 days. Media was then removed and cells were fixed with 70% v/vmethanol for 1 minute. Cells were then stained with 0.2% w/v crystalviolet in 70% v/v ethanol for 10 seconds, washed with dH₂O and allowedto air dry. The number of colonies formed of >50 cells each werecounted.

Drugs were used in the following concentrations for Growth inhibitionand Clonogenic assays: Geldanamycin   1-1500 nM Irinotecan 0.01-100 μMRadicicol   25-350 nM Topotecan 12.5-800 nM2.1.3 Flow Cytometry Protocol for Cell Cycle Analysis.

-   1. Seed cells eg HCT116+/+ or K562 cells in small petri dish or 6    well plate using 5 ml of 1×10⁶ cells/ml in appropriate medium. For    HCT116+/+ cell line use McCoys 5A Medium supplemented with 10%    Foetal Calf Serum (FCS) and Penicillin and Streptomycin. For K562    cell line use RPMI 1640 Medium supplemented with 10% FCS and    Penicillin and Streptomycin.-   2. Leave to attach overnight for adherent cell lines in incubator at    37° C. 5% CO₂ atmosphere.*    * For suspension cell lines, spin cells down and resuspend at    between 2-4×10⁵ cells/ml in medium supplemented with the drug    treatment required.-   3. Dose with 5 ml of drug/control for required time course in    incubator at 37° C. 5% CO₂ atmosphere. 125 nM Geldanamycin, 0.5 μM    Irinotecan, or 125 nM Geldanamycin and 0.5 μM Irinotecan    combination.-   4. After treatment, remove medium from well to a universal tube.**    ** For suspension cell lines ignore steps 6 and 7.-   5. Wash well with 500 μl PBS and remove to same universal.-   6. Add 500 μl trypsin and wait for detachment.-   7. Add trypsin and cells to universal and rinse out the well with    some of the medium from the universal.-   8. Spin cells at 4° C. at 2500 rpm for 5 mins.-   9. Remove supernatant and resuspend pellet in 500 μl PBS-   10. Transfer to Falcon tube and spin at 4° C. at 2500 rpm for 5    minutes-   11. Remove supernatant and add 500 μl ice-cold 70% ethanol, and    leave in fridge for 2-5 minutes.-   12. Spin cells at 4° C. at 2500 rpm for 5 mins.-   13. Wash twice in 1 ml PBS.-   14. Add 40 μl of 100 μg/ml ribonuclease A for 5 mins at room    temperature.-   15. Add 400 μl of 50 μg/ml propidium iodide (Sigma) and incubate for    15 minutes.-   16. Analyse on FACSVantage SE (Becton Dickinson) using 488 nm laser    for excitation, and collecting fluorescence above 585 nm (FL-2).    Collect data using CellQuest Pro v4.0. Analyse data using Mod Fit LT    v3.0    2.1.4 Isobolar Relations

The isobolar relations were calculated to quantify the synergisticcombination of the two agents used according to the invention.

The isobolar relation is calculated in the light of the fact that twodrugs used in combination may produce enhanced or reduced effects. Thedegree of enhancement or reduction is measured from the interactionindex (γ), defined by the isobolar relation, which indicates the changedpotency of the combination.(a/A)+(b/B)=γWhere:A=drug A alone; B=drug B alone; and a, b=combination dose toproduce desired effect

If γ 1 =additive; <1=super-additive (synergistic); and >1=sub-additive

2.2 Results

2.2.1 Growth Inhibition Assay

Example 1 illustrates that the interaction between these two proteinsrepresents a new drug target. The effectiveness of modulating the twoproteins in chemotherapy was tested using inhibitors of HSP 90 andinhibitors of Topo I in combination.

The beneficial effects of inhibitors of HSP 90 and inhibitors of Topo Iare shown in FIGS. 4-9.

The inhibitory effects of Irinotecan alone or Geldanamycin alone on cellgrowth are shown in FIG. 4 and FIG. 5, respectively. Moreover, thesynergistic inhibitory effect of the combination of Irinotecan andGeldanamycin on cell proliferation is shown in FIGS. 6 and 7.

Synergistic effects were also seen when Radicicol was combined withIrinotecan (see FIG. 8) and when Topotecan was combined withGeldanamycin (see FIG. 9).

2.2.2 Clonogenic Assay

In the clonogenic assays, cell killing, showed a similar synergisticeffect when a combination of HSP 90 and Topo I inhibitors was used. Thecombination of Irinotecan and Geldanamycin resulted in less colonysurvival than treatment with single drugs (FIG. 10).

In FIG. 11, the cell killing response of HCT116 p53 cells aftertreatment with two agents is shown. Overall, this produced a synergisticeffect and resulted in cell killing at concentrations, where there islittle or no effect with single drugs. Thus, cell killing was found tobe at least 3-5 times greater fro combination therapy than for the drugused in isolation. The action was also found to be independent of p53status.

Flow cytometry experiments indicate that greater DNA damage may be seenwith combination therapy according to the invention.

2.2.3 Isobolar Relations

FIG. 12 shows an isobologram illustrating the cell killing responseafter treatment with irinotecan, geldanamycin andirinotecan/geldanamycin in combination on (A) HCT116 p53 WT cells and(B) p53 KO cells as described in Example 2; and

It will therefore be appreciated that the combination of the two agentsproduces a synergistic effect (i.e. cell killing at concentrations,where there is little or no effect with single drugs)

EXAMPLE 3

Experiments were conducted to illustrate the efficacy of the combinationtherapy according to the present invention for destroyingmircroorganisms

3.1 Methods

An E.coli extract was generated from a 50 ml over night culture ofbacteria (shaking culture at 37° C.). The culture was harvested andresuspended in lysis buffer 0.5 ml and sonicated to disrupt the cells,the extract was then cleared of debris by centrifugation at 12,000 g for15 minutes the cleared extract was then used for the immunoprecipitation(all these procedures were carried out at 4° C.).

Co-immunoprecipitation (IP) western blots of IPs from E.coli extractswere then carried out. Initially, immunoprecipitation with ananti-topoisomerase I antibody the western blot was probed with ananti-body against Hsp90 (which recognises the bacterial equivalentHtpG). This was accompanied by an IP with anti Hsp90 antibody (whichrecognises the bacterial equivalent HtpG) whose western blot was probedwith a topoisomerase I antibody.

3.2 Results

This example demonstrates that HSP90 and Topoisomerase I interact andinfluence microbial cell growth and susceptibility to chemotherapy.

FIG. 13 shows western blots of immunoprecipitation and counterimmunoprecipitation probed with the complementary antibodies,demonstrating that the corresponding proteins come down in pull downexperiments and thereby illustrating that Hsp90 and Topoisomerase Ihomologues in bacteria interact and influence cell growth and death.

1. A method of administering chemotherapy, comprising: administering afirst agent that attenuates Topoisomerase I (Topo I) activity; andadministering a second agent that inhibits Heat Shock Protein 90 (HSP90)activity, wherein the first agent and the second agent are administeredeither contemporaneously or sequentially.
 2. The method of claim 1wherein the first agent is a compound selected from the group consistingof: (i) compounds that bind to Topo I and inhibit its activity, (ii)compounds which prevent the transcription, translation or expression ofTopo I, (iii) compounds which inhibit release of Topo I fromintracellular stores, and (iv) compounds which increase the rate ofdegradation of Topo I.
 3. The method of claim 1 wherein the first agentis a cleavable-complex inhibitor.
 4. The method of claim 1 wherein thefirst agent is Camptothecin.
 5. The method of claim 1 wherein the firstagent is Topotecan.
 6. The method of claim 1 wherein the first agent isIrinotecan.
 7. The method of claim 1 wherein the first agent isCamptostar (CPT-11).
 8. The method of claim 1 wherein the first agent isGemcitabine.
 9. The method of claim 1 wherein the second agent is acompound selected from the group consisting of: (i) compounds that bindto HSP 90 and inhibit its activity, (ii) compounds which prevent thetranscription, translation or expression of HSP 90, (iii) compoundswhich inhibit release of HSP 90 from intracellular stores, and (iv)compounds which increase the rate of degradation of HSP
 90. 10. Themethod of claim 9 wherein the second agent is Geldanamycin.
 11. Themethod of claim 10 wherein the second agent is 17-Allylamino,17-demethoxygeldanamycin (17AAG) or CNF-101.
 12. The method of claim 9wherein the second agent is Radicicol.
 13. The method of claim 1 whereinthe chemotherapy is for cancer treatment.
 14. The method of claim 13 forthe treatment of solid tumours.
 15. The method of claim 14 for thetreatment of bowel cancer, small cell and non-small cell lung cancer,head and neck cancer, breast cancer, bladder cancer or malignantmelanoma.
 16. The method of claim 15 for the treatment of paediatrictumours.
 17. The method of claim 13 for the treatment of neuroblastoma,leukaemias and/or lymphomas.
 18. The method of claim 1 wherein thechemotherapy is for: antibacterial treatments, antifungal treatments,antiparasitic treatments, the treatment of AIDS/HIV, the treatment ofmultiple sclerosis, or the killing and inhibition of proliferation ofany organism.
 19. The method of claim 1 wherein the chemotherapy is forprophylactic treatment.